Detection of markers in nascent proteins

ABSTRACT

The invention is directed to methods for the non-radioactive labeling, detection, quantitation and isolation of nascent proteins translated in a cellular or cell-free translation system. tRNA molecules are misaminoacylated with non-radioactive markers which may be non-native amino acids, amino acid analogs or derivatives, or substances recognized by the protein synthesizing machinery. Markers may comprise cleavable moieties, detectable labels, reporter properties wherein markers incorporated into protein can be distinguished from unincorporated markers, or coupling agents which facilitate the detection and isolation of nascent,protein from other components of the translation system. The invention also comprises proteins prepared using misaminoacylated tRNAs which can be utilized in pharmaceutical compositions for the treatment of diseases and disorders in humans and other mammals, and kits which may be used for the detection of diseases and disorders.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to non-radioactive markers that facilitate thedetection of nascent proteins translated within cellular or cell-freetranslation systems Nascent proteins containing these markers can berapidly and efficiently isolated without the handling and disposalproblems associated with radioactive reagents.

2. Description of the Background

Cells contain organelles, macromolecules and a wide variety of smallmolecules. Except for water, the vast majority of the molecules andmacromolecules can be classified as lipids, carbohydrates, proteins ornucleic acids. Proteins are the most abundant cellular components andfacilitate many of the key cellular processes. They include enzymes,antibodies, hormones, transport molecules and components for thecytoskeleton of the cell.

Proteins are composed of amino acids arranged into linear polymers orpolypeptides. In Living systems, proteins comprise over twenty commonamino acids. These twenty or so amino acids are generally termed thenative amino acids. At the center of every amino acid is the alphacarbon atom (Cα) which forms four bonds or attachments with othermolecules (FIG. 1A). One bond is a covalent linkage to an amino group(NH₂) and another to a carboxyl group (COOH) which both participate inpolypeptide formation A third bond is nearly always linked to a hydrogenatom and the fourth to a side chain which imparts variability to theamino acid structure. For example, alanine is formed when the side chainis a methyl group (—CH₃) and a valine is formed when the side chain isan isopropyl group (—CH(CH₃)₂). It is also possible to chemicallysynthesize amino acids containing different side-chains, however, thecellular protein synthesis system, with rare exceptions, utilizesnative-amino acids. Other amino acids and structurally similar chemicalcompounds are termed non-native and are generally not found in mostorganisms.

A central feature of all living systems is the ability to produceproteins from amino acids. Basically, protein is formed by the linkageof multiple amino acids via peptide bonds (—CO—H—) such as thepentapeptide depicted in FIG. 1B. Key molecules involved in this processare messenger RNA (mRNA) molecules, transfer RNA (tRNA) molecules andribosomes (rRNA-protein complexes). Protein translation normally occursin living cells and in some cases can also be performed outside the cellin systems referred to as cell-free translation systems. In eithersystem, the basic process of protein synthesis is identical. Theextra-cellular or cell-free translation system comprises an extractprepared from the intracellular contents of cells. These preparationscontain those molecules which support protein translation and dependingon the method of preparation, post-translational events such asglycosylation and cleavages as well. Typical cells from which cell-freeextracts or in vitro extracts are made are Escherichia coli cells, wheatgerm cells, rabbit reticulocytes, insect cells and frog oocytes.

Both in vivo and in vitro syntheses involve the reading of a sequence ofbases on a mRNA molecule. The mRNA contains instructions for translationin the form of triplet codons. The genetic code specifies which aminoacid is encoded by each triplet codon. For each codon which specifies anamino acid, there normally exists a cognate tRNA molecule whichfunctions to transfer the correct amino acid onto the nascentpolypeptide chain. The amino acid tyrosine (Tyr) is coded by thesequence of bases UAU and UAC, while cysteine (Cys) is coded by UGU andUGC. Variability associated with the third base of the codon is commonand is called wobble.

Translation begins with the binding of the ribosome to mRNA (FIG. 2). Anumber of protein factors associate with the ribosome during differentphases of translation including initiation factors, elongation factorsand termination factors. Formation of the initiation complex is thefirst step of translation Initiation factors contribute to theinitiation complex along with the mRNA and initiator tRNA (fmet and met)which recognizes the base sequence UAG. Elongation proceeds with chargedtRNAs binding to ribosomes, translocation and release of the amino acidcargo into the peptide chair Elongation factors assist with the bindingof tRNAs and in elongation of the polypeptide chain with the help ofenzymes like peptidyl transferase. Termination factors recognize a stopsignal, such as the base sequence UGA, in the message terminatingpolypeptide synthesis and releasing the polypeptide chain and the mRNAfrom the ribosome.

The structure of tRNA is often shown as a cloverleaf representation(FIG. 3A). Structural elements of a typical tRNA include an acceptorstem, a D-loop, an anticodon loop, a variable loop and a TΨC loop.Aminoacylation or charging of tRNA results in linking the carboxylterminal of an amino acid to the 2′-(or 3′-) hydroxyl group of aterminal adenosine base via an ester linkage. This process can beaccomplished either using enzymatic or chemical methods. Normally aparticular tRNA is charged by only one specific native amino acid. Thisselective charging, termed here enzymatic aminoacylation, isaccomplished by aminoacyl tRNA synthetases. A tRNA which selectivelyincorporates a tyrosine residue into the nascent polypeptide chain byrecognizing the tyrosine UAC codon will be charged by tyrosine with atyrosine-aminoacyl tRNA synthetase, while a tRNA designed to read theUGU codon will be charged by a cysteine-aminoacyl tRNA synthetase. Thesesynthetases have evolved to be extremely accurate in charging a tRNAwith the correct amino acid to maintain the fidelity of the translationprocess. Except in special cases where the non-native amino acid is verysimilar structurally to the native amino acid, it is necessary to usemeans other than enzymatic aminoacylation to charge a tRNA.

Molecular biologists routinely study the expression of proteins that arecoded for by genes. A key step in research is to express the products ofthese genes either in intact cells or in cell-free extracts.Conventionally, molecular biologists use radioactively labeled aminoacid residues such as ³⁵S-methionine as a means of detecting newlysynthesized proteins or so-called nascent proteins. These nascentproteins can normally be distinguished from the many other proteinspresent in a cell or a cell-free extract by first separating theproteins by the standard technique of gel electrophoresis anddetermining if the proteins contained in the gel possess the specificradioactively labeled amino acids. This method is simple and relies ongel electrophoresis, a widely available and practiced method. It doesnot require prior knowledge of the expressed protein and in general doesnot require the protein to have any special properties. In addition, theprotein can exist in a denatured or unfolded form for detection by gelelectrophoresis. Furthermore, more specialized techniques such asblotting to membranes and coupled enzymatic assays are not needed.Radioactive assays also have the advantage that the structure of thenascent protein is not altered or can be restored, and thus, proteinscan be isolated in a functional form for subsequent biochemical andbiophysical studies.

Radioactive methods suffer from many drawbacks related to theutilization of radioactively labeled amino acids. Handling radioactivecompounds in the laboratory always involves a health risk and requiresspecial laboratory safety procedures, facilities and detailed recordkeeping as well as special training of laboratory personnel. Disposal ofradioactive waste is also of increasing concern both because of thepotential risk to the public and the lack of radioactive waste disposalsites. In addition, the use of radioactive labeling is time consuming,in some cases requiring as much as several days for detection of theradioactive label. The long time needed for such experiments is a keyconsideration and can seriously impede research productivity. Whilefaster methods of radioactive detection are available, they areexpensive and often require complex image enhancement devices.

The use of radioactive labeled amino acids also does not allow for asimple and rapid means to monitor the production of nascent proteinsinside a cell-free extract without prior separation of nascent frompreexisting proteins. However, a separation step does not allow for theoptimization of cell-free activity. Variables including theconcentration of ions and metabolites and the temperature and the timeof protein synthesis cannot be adjusted.

Radioactive labeling methods also do not provide a means of isolatingnascent proteins in a form which can be further utilized. The presenceof radioactivity compromises this utility for further biochemical orbiophysical procedures in the laboratory and in animals. This is clearin the case of in vitro expression when proteins cannot be readilyproduced in vivo because the protein has properties which are toxic tothe cell. A simple and convenient method for the detection and isolationof nascent proteins in a functional form could be important in thebiomedical field if such proteins possessed diagnostic or therapeuticproperties. Recent research has met with some success, but these methodshave had numerous drawbacks.

Special tRNAs, such as tRNAs which have suppressor properties,suppressor tRNAs, have been used in the process of site-directednon-native amino acid replacement (SNAAR) (C. Noren et al., Science244:182-188, 1989). In SNAAR, a unique codon is required on the mRNA andthe suppressor tRNA, acting to target a non-native amino acid to aunique site during the protein synthesis (PCT WO90/05785). However, thesuppressor tRNA must not be recognizable by the aminoacyl tRNAsynthetases present in the protein translation system (Bain et al.,Biochemistry 30:5411-21, 1991). Furthermore, site-specific incorporationof non-native amino acids is not suitable in general for detection ofnascent proteins in a cellular or cell-free protein synthesis system dueto the necessity of incorporating non-sense codons into the codingregions of the template DNA or the mRNA.

Products of protein synthesis may also be detected by using antibodybased assays. This method is of limited use because it requires that theprotein be folded into a native form and also for antibodies to havebeen previously produced against the nascent protein or a known proteinwhich is fused to the unknown nascent protein. Such procedures are timeconsuming and again require identification and characterization of theprotein. In addition, the production of antibodies and amino acidsequencing both require a high level of protein purity.

In certain cases, a non-native amino acid can be formed after the tRNAmolecule is aminoacylated using chemical reactions which specificallymodify the native amino acid and do not significantly alter thefunctional activity of the aminoacylated tRNA (Promega TechnicalBulletin No. 182; tRNA^(nscend ™): Non-radioactive Translation DetectionSystem, September 1993). These reactions are referred to aspost-aminoacylation modifications. For example, the ε-amino group of thelysine linked to its cognate tRNA (tRNA^(LYS)), could be modified withan amine specific photoaffinity label (U. C. Krieg et al., Proc. Natl.Acad. Sci. USA 83:8604-08, 1986). These types of post-aminoacylationmodifications, although useful, do not provide a general means ofincorporating non-native amino acids into the nascent proteins. Thedisadvantage is that only those non-native amino acids that arederivatives of normal amino acids can be incorporated and only a fewamino acid residues have side chains amenable to chemical modification.More often, post-aminoacylation modifications can result in the tRNAbeing altered and produce a non-specific modification of the α-aminogroup of the amino acid (e.g. in addition to the ε-amino group) linkedto the tRNA. This factor can lower the efficiency of incorporation ofthe non-native amino acid linked to the tRNA. Non-specific,post-aminoacylation modifications of tRNA structure could alsocompromise its participation in protein synthesis. Incomplete chainformation could also occur when the α-amino group of the amino acid ismodified.

In certain other cases, a nascent protein can be detected because of itsspecial and unique properties such as specific enzymatic activity,absorption or fluorescence. This approach is of limited use since mostproteins do not have special properties with which they can be easilydetected. In many cases, however, the expressed protein may not havebeen previously characterized or even identified, and thus, itscharacteristic properties are unknown.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides methods forthe labeling, detection, quantitation and isolation of nascent proteinsproduced in a cell-free or cellular translation system without the useof radioactive amino acids or other radioactive labels.

One embodiment of the invention is directed to methods for detectingnascent proteins translated in a translation system. A tRNA molecule isaminoacylated with a cleavable marker to create a misaminoacylated tRNA.The misaminoacylated, or charged, tRNA can be formed by chemical,enzymatic or partly chemical and partly enzymatic techniques which placea cleavable marker into a position on the tRNA molecule from which itcan be transferred into a growing peptide chain. Cleavable markers maycomprise native amino acids with cleavable moieties, non-native aminoacids, amino acid analogs or derivatives, detectable labels, couplingagents or combinations of these components. The misaminoacylated tRNA isintroduced to the translation system such as a cell-free extract, thesystem is incubated and marker incorporated into nascent proteins. Thesemethods may be used to detect, isolate and quantitate such nascentproteins as recombinant gene products, gene fusion products, enzymes,cytokines, hormones, immunogenic proteins, human proteins, carbohydrateand lipid binding proteins, nucleic acid binding proteins, viralproteins, bacterial proteins, parasitic proteins and fragments andcombinations thereof.

Another embodiment of the invention is directed to methods for labelingnascent proteins at their amino terminus. An initiator tRNA molecule,such as methionine-initiator tRNA or formylmethionine-initiator tRNA ismisaminoacylated as before and introduced to a translation system. Thesystem is incubated and marker is incorporate at the amino terminus ofthe nascent proteins. Nascent proteins containing marker can bedetected, isolated and quantitated. Markers or parts of markers may becleaved from the nascent proteins which substantially retain theirnative configuration and are functionally active.

Another embodiment of the invention is directed to methods for thedetection of nascent proteins translated in a cellular or cell-freetranslation system using non-radioactive markers which have detectableelectromagnetic spectral properties. As before, a non-radioactive markeris misaminoacylated to a tRNA molecule and the misaminoacylated tRNA isadded to the translation system. The system is incubated to incorporatemarker into the nascent proteins. Nascent proteins containing marker canbe detected from the specific electromagnetic spectral property of themarker. Nascent proteins can also be isolated by taking advantage ofunique properties of these markers or by conventional means such aselectrophoresis, gel filtration, high-pressure or fast-pressure liquidchromatography, affinity chromatography, ion exchange chromatography,chemical extraction, magnetic bead separation, precipitation orcombinations of these techniques.

Another embodiment of the invention is directed to the synthesis ofnascent proteins containing markers which have reporter properties.Reporter markers are chemical moieties which have detectableelectromagnetic spectral properties when incorporated into peptides andwhose spectral properties can be distinguished from unincorporatedmarkers and markers attached to tRNA molecules. As before, tRNAmolecules are misaminoacylated, this time using reported markers. Themisaminoacylated tRNAs are added to a translation system and incubatedto incorporate marker into the peptide. Reporter markers can be used tofollow the process of protein translation and to detect and quantitatenascent proteins without prior isolation from other components of theprotein synthesizing system.

Another embodiment of the invention is directed to compositionscomprised of nascent proteins translated in the presence of markers,isolated and, if necessary, purified in a cellular or cell-freetranslation system. Compositions may further comprise a pharmaceuticallyacceptable carrier and be utilized as an immunologically activecomposition such as a vaccine, or as a pharmaceutically activecomposition such as a drug, for use in humans and other mammals.

Other embodiments and advantages of the invention are set forth, inpart, in the description which follows and, in part will be obvious fromthis description, or may be learned from the practice of the invention.

DESCRIPTION OF THE DRAWINGS

FIG. 1 Structure of (A) an amino acid and (B) a peptide.

FIG. 2 Description of the molecular steps that occur during proteinsynthesis in a cellular or cell-free system.

FIG. 3 Structure of (A) a tRNA molecule and (B) approaches involved inthe aminoacylation of tRNAs.

FIG. 4 Schematic representation of the method of detecting nascentproteins using fluorescent marker amino acids.

FIG. 5 Schemes for synthesis and misaminoacylation to tRNA of twodifferent marker amino acids, dansyllysine (scheme 1) and coumarin(scheme 2), with fluorescent properties suitable for the detection ofnascent proteins using gel electrophoresis and UV illumination.

FIG. 6 (A) Chemical compounds containing the 2-nitrobenzyl moiety, and(B) cleavage of substrate from a nitrobenzyl linkage.

FIG. 7 Examples of photocleavable markers.

FIG. 8 (A) Chemical variations of PCB, and (B) possible amino acidlinkages.

FIG. 9 Photolysis of PCB.

FIG. 10 Schematic representation of the method for monitoring theproduction of nascent proteins in a cell-free protein expression systemswithout separating the proteins.

FIG. 11 (A) Examples of non-native amino acids with reporter properties,(B) participation of a reporter in protein synthesis, and (C) synthesisof a reporter.

FIG. 12 Structural components of photocleavable biotin.

FIG. 13 Schematic representation of the method for introduction ofmarkers at the N-termini of nascent proteins.

FIG. 14 Description of the method of detection and isolation of markerin nascent proteins.

FIG. 15 Steps in the synthesis of PCB-lysine.

FIG. 16 Experimental strategy for the misaminoacylation of tRNA.

FIG. 17 Dinucleotide synthesis including (i) deoxycytidine protection,(ii) adenosine protection, and (iii) dinucleotide synthesis.

FIG. 18 Aminoacylation of a dinucleotide using marker amino acids.

DESCRIPTION OF THE INVENTION

As embodied and broadly described herein, the present inventioncomprises methods for the non-radioactive labeling and detection of theproducts of new or nascent protein synthesis, and methods for theisolation of these nascent proteins from preexisting proteins in acellular or cell-free translation system As radioactive labels are notused, there are no special measures which must be taken to dispose ofwaste materials. There is also no radioactivity danger or risk whichwould prevent further utilization of the translation product as occurswhen using radioactive labels and the resulting protein product may beused directly or further purified. In addition, no prior knowledge ofthe protein sequence or structure is required which would involve, forexample, unique suppressor tRNAs. Further, the sequence of the gene ormRNA need not be determined. Consequently, the existence of non-sensecodons or any specific codons in the coding region of the mRNA is notnecessary. Any tRNA can be used, including specific tRNAs for directedlabeling, but such specificity is not required. Unlikepost-translational labeling, nascent proteins are labeled withspecificity and without being subjected to post-translationalmodifications which may effect protein structure or function.

One embodiment of the invention is directed to a method for labelingnascent proteins synthesized in a translation system. These proteins arelabeled while being synthesized with detectable markers which areincorporated into the peptide chain. Markers which are aminoacylated totRNA molecules, may comprise native amino acids, non-native amino acids,amino acid analogs or derivatives, or chemical moieties. These markersare introduced into nascent proteins from the resulting misaminoacylatedtRNAs during the translation process. Aminoacylation is the processwhereby a tRNA molecule becomes charged. When this process occurs invivo, it is referred to as natural aminoacylation and the resultingproduct is an aminoacylated tRNA charged with a native amino acid. Whenthis process occurs through artificial means, it is calledmisaminoacylation and a tRNA charged with anything but a native aminoacid molecule is referred to as a misaminoacylated tRNA.

According to the present method, misaminoacylated tRNAs are introducedinto a cellular or cell-free protein synthesizing system, thetranslation system, where they function in protein synthesis toincorporate detectable marker in place of a native amino acid in thegrowing peptide chain. The translation system comprises macromoleculesincluding RNA and enzymes, translation, initiation and elongationfactors, and chemical reagents. RNA of the system is required in threemolecular forms, ribosomal RNA (rRNA), messenger RNA (mRNA) and transferRNA (tRNA). mRNA carries the genetic instructions for building a peptideencoded within its codon sequence. tRNAs contain specific anti-codonswhich decode the mRNA and individually carry amino acids into positionalong the growing peptide chum Ribosomes, complexes of rRNA and protein,provide a dynamic structural framework on which the translation process,including translocation, can proceed. Within the cell individualizedaminoacyl tRNA synthetases bind specific amino acids to tRNA moleculescarrying the matching anti-codon creating aminoacylated or charged tRNAsby the process of aminoacylation. The process of translation includingthe aminoacylation or charging of a tRNA molecule is described inMolecular Cell Biology (J. Darnell et al. editors, Scientific AmericanBooks, N.Y., N.Y. 1991), which is hereby specifically incorporated byreference.

Aminoacylation may be natural or by artificial means utilizing nativeamino acids, non-native amino acid, amino acid analogs or derivatives,or other molecules such as detectable chemicals or coupling agents. Theresulting misaminoacylated tRNA comprises a native amino acid coupledwith a chemical moiety, non-native amino acid, amino acid derivative oranalog, or other detectable chemicals. These misaminoacylated tRNAsincorporate their markers into the growing peptide chain duringtranslation forming labeled nascent proteins which can be detected andisolated by the presence or absence of the marker.

Any proteins that can be expressed by translation in a cellular orcell-free translation system may be nascent proteins and consequently,labeled, detected and isolated by the methods of the invention Examplesof such proteins include enzymes such as proteolytic proteins,cytokines, hormones, immunogenic proteins, carbohydrate or lipid bindingproteins, nucleic acid binding proteins, human proteins, viralproteins, * bacterial proteins, parasitic proteins and fragments andcombinations. These methods are well adapted for the detection ofproducts of recombinant genes and gene fusion products becauserecombinant vectors carrying such genes generally carry strong promoterswhich transcribe mRNAs at fairly high levels. These mRNAs are easilytranslated in a translation system.

Translation systems may be cellular or cell-free, and may be prokaryoticor eukaryotic. Cellular translation systems include whole cellpreparations such as permeabilized cells or cell cultures wherein adesired nucleic acid sequence can be transcribed to mRNA and the mRNAtranslated.

Cell-free translation systems are commercially available and manydifferent types and systems are well-known Examples of cell-free systemsinclude prokaryotic lysates such as Escherichia coli lysates, andeukaryotic lysates such as wheat germ extracts, insect cell lysates,rabbit reticulocyte lysates, rabbit oocyte lysates and human celllysates. Eukaryotic extracts or lysates may be preferred when theresulting protein is glycosylated, phosphorylated or otherwise modifiedbecause many such modifications are only possible in eukaryotic systems.Some of these extracts and lysates are available commercially (Promega;Madison, Wis.; Stratagene; La Jolla, Calif.; Amersham; ArlingtonHeights, Ill.; GIBCO/BRL; Grand Island, N.Y.). Membranous extracts, suchas the canine pancreatic extracts containing microsomal membranes, arealso available which are useful for translating secretory proteins.Mixtures of purified translation factors have also been usedsuccessfully to translate mRNA into protein as well as combinations oflysates or lysates supplemented with purified translation factors suchas initiation factor-1 (IF-1), LF-2, IF-3 (α or β), elongation factor T(EF-Tu), or termination factors.

Cell-free systems may also be coupled transcription/translation systemswherein DNA is introduced to the system, transcribed into mRNA and themRNA translated as described in Current Protocols in Molecular Biology(F. M. Ausubel et al. editors, Wiley Interscience, 1993), which ishereby specifically incorporated by reference. RNA transcribed ineukaryotic transcription system may be in the form of heteronuclear RNA(mRNA) or 5′-end caps (7-methyl guanosine) and 3′-end poly A tailedmature mRNA, which can be an advantage in certain translation systems.For example, capped mRNAs are translated with high efficiency in thereticulocyte lysate system.

tRNA molecules chosen for misaminoacylation with marker do notnecessarily possess any special properties other than the ability tofunction in the protein synthesis system. Due to the universality of theprotein translation system in living systems, a large number of tRNAscan be used with both cellular and cell-free reaction mixtures. SpecifictRNA molecules which recognize unique codons, such as nonsense or ambercodons (UAG), are not required.

Site-directed incorporation of the nonnative analogs into the proteinduring translation is also not required. Incorporation of markers canoccur anywhere in the polypeptide and can also occur at multiplelocations. This eliminates the need for prior information about thegenetic sequence of the translated mRNA or the need for modifying thisgenetic sequence.

In some cases, it may be desirable to preserve the functional propertiesof the nascent protein. A subset of tRNAs which will incorporate markersat sites which do not interfere with protein function or structure canbe chosen. Amino acids at the amino or carboxyl terminus of apolypeptide do not alter significantly the function or structure. tRNAmolecules which recognize the universal codon for the initiation ofprotein translation (AUG), when misaminoacylated with marker, will placemarker at the amino terminus. Prokaryotic protein synthesizing systemsutilize initiator tRNA^(fMet) molecules and eukaryotic systems initiatortRNA^(Met) molecules. In either system the initiator tRNA molecules areaminoacylated with markers which may be non-native amino acids or aminoacid analogs or derivatives that possess marker, reporter or affinityproperties. The resulting nascent proteins will be exclusively labeledat their amino terminus, although markers placed internally do notnecessarily destroy structural or functional aspects of a protein. Forexample, a tRNA^(LYS) may be misaminoacylated with the amino acidderivative dansyllysine which does not interfere with protein functionor structure. In addition, using limiting amounts of misaminoacylatedtRNAs, it is possible to detect and isolate nascent proteins having onlya very small fraction labeled with marker which can be very useful forisolating-proteins when the effects of large quantities of marker wouldbe detrimental or are unknown.

tRNAs molecules used for aminoacylation are commercially available froma number of sources and can be prepared using well-known methods fromsources including Escherichia coli, yeast, calf liver and wheat germcells (Sigma Chemical; St. Louis, Mo.; Promega; Madison, Wis.;Boehringer Mannheim Biochemicals; Indianapolis, Ind.). Their isolationand purification mainly involves cell-lysis, phenol extraction followedby chromatography on DEAE-cellulose. Amino-acid specific tRNA, forexample tRNA^(fMet), can be isolated by expression from cloned genes andoverexpressed in host cells and separated from total tRNA by techniquessuch as preparative polyacrylamide gel electrophoresis followed by bandexcision and elution in high yield and purity (Seong and RajBhandary,Proc. Natl. Acad. Sci. USA.84:334-338, 1987). Run-off transcriptionallows for the production of any specific tRNA in high purity, but itsapplications can be limited due to lack of post-transcriptionalmodifications (Bruce and Uhlenbeck, Biochemistry 21:3921, 1982).

Misaminoacylated tRNAs are introduced into the cellular or cell-freeprotein synthesis system. In the cell-free protein synthesis system, thereaction mixture contains all the cellular components necessary tosupport protein synthesis including ribosomes, tRNA, rRNA, spermidineand physiological ions such as magnesium and potassium at appropriateconcentrations and an appropriate pH. Reaction mixtures can be normallyderived from a number of different sources including wheat germ, E. coli(S-30), red blood-cells (reticulocyte lysate,) and oocytes, and oncecreated can be stored as aliquots at about +4° C. to −70° C. The methodof preparing such reaction mixtures is described by J. M. Pratt(Transcription and Translation, B. D. Hames and S. J. Higgins Editors,p. 209, LRL Press, Oxford, 1984) which is hereby incorporated byreference. Many different translation systems are commercially availablefrom a number of manufacturers.

The misaminoacylated tRNA is added directly to the reaction mixture as asolution of predetermined volume and concentration. This can be donedirectly prior to storing the reaction mixture at −70° C. in which casethe entire mixture is thawed prior to initiation of protein synthesis orprior to the initiation of protein synthesis. Efficient incorporation ofmarkers into nascent proteins is sensitive to the final pH and magnesiumion concentration. Reaction mixtures are normally about pH 6.8 andcontain a magnesium ion concentration of about 3 mM. These conditionsimpart stability to the base-labile aminoacyl linkage of themisaminoacylated tRNA. Aminoacylated tRNAs are available in sufficientquantities from the translation extract. Misaminoacylated tRNAs chargedwith markers are added at between about 1.0 μm /ml to about 1.0 mg/ml,preferably at between about 10 μg/ml to about 500 μg/ml, and morepreferably at about 150 μg/ml.

Initiation of protein synthesis occurs upon addition of a quantity ofmRNA or DNA to the reaction mixture containing the misaminoacylatedtRNAs. mRNA molecules may be prepared or obtained from recombinantsources, or purified from other cells by procedure such as poly-dTchromatography. One method of assuring that the proper ratio of thereaction mixture components is to use predetermined volumes that arestored in convenient containers such as vials or standardmicrocentrifuge tubes. For example, DNA and/or mRNA coding for thenascent proteins and the misaminoacylated tRNA solution are premixed inproper amounts and stored separately in tubes. Tubes are mixed whenneeded to initiate protein synthesis.

Translations in cell-free systems generally require incubation of theingredients for a period of time. Incubation times range from about 5minutes to many hours, but is preferably between about thirty minutes toabout five hours and more preferably between about one to about threehours. Incubation may also be performed in a continuous manner wherebyreagents are flowed into the system and nascent proteins removed or leftto accumulate using a continuous flow system (A. S. Spirin et al., Sci.242:1162-64, 1988). This process may be desirable for large scaleproduction of nascent proteins. Incubation times vary significantly withthe volume of the translation mix and the temperature of the incubation.Incubation temperatures can be between about 4° C. to about 60° C., andare preferably between about 15° C. to about 50° C., and more preferablybetween about 25° C. to about 45° C. and even more preferably at about25° C. or about 37° C. Certain markers may be sensitive to temperaturefluctuations and in such cases, it is preferable to conduct thoseincubations in the non-sensitive ranges. Translation mixes willtypically comprise buffers such as Tris-HCl, Hepes or another suitablebuffering agent to maintain the pH of the solution between about 6 to 8,and preferably at about 7. Again, certain markers may be pH sensitiveand in such cases, it is preferable to conduct incubations outside ofthe sensitive ranges for the marker. Translation efficiency may not beoptimal, but marker utility will be enhanced. Other reagents which maybe in the translation system include dithiothreitol (DTT) or2-mercaptoethanol as reducing agents, RNasin to inhibit RNA breakdown,and nucleoside triphosphates or creatine phosphate and creatine kinaseto provide chemical energy for the translation process.

In cellular protein synthesis, it is necessary to introducemisaminoacylated tRNAs or markers into intact cells, cell organelles,cell envelopes and other discrete volumes bounded by an intactbiological membrane, which contain a protein synthesizing system. Thiscan be accomplished through a variety of methods that have beenpreviously established such as sealing the tRNA solution into liposomesor vesicles which have the characteristic that they can be induced tofuse with cells. Fusion introduces the liposome or vesicle interiorsolution containing the tRNA into the cell. Alternatively, some cellswill actively incorporate liposomes into their interior cytoplasmthrough phagocytosis. The tRNA solution could also be introduced throughthe process of cationic detergent mediated lipofection (Feigner et al.,Proc. Natl. Acad. Sci. USA 84:7413-17, 1987), or injected into largecells such as oocytes. Injection may be through direct perfusion withmicropipettes or through the method of electroporation.

Alternatively, cells can be permeabilized by incubation for a shortperiod of time in a solution containing low concentrations of detergentsin a hypotonic media.

Useful detergents include Nonidet-P 40 (NP40), Triton X-100 (TX-100) ordeoxycholate at concentrations of about 0.01 nM to 1.0 mM, preferablybetween about 0.1 μM to about 0.01 mM, and more preferably about 1 μM.Permeabilized cells allow marker to pass through cellular membranesunaltered and be incorporated into nascent proteins by host cellenzymes. Such systems can be formed from intact cells in culture such asbacterial cells, primary cells, immortalized cell lines, human cells ormixed cell populations. These cells may, for example, be transfectedwith an appropriate vector containing the gene of interest, under thecontrol of a strong and possibly regulated promoter. Messages areexpressed from these vectors and subsequently translated within cells.Intact misaminoacylated tRNA molecules, already charged with anon-radioactive marker could be introduced to cells and incorporatedinto translated product.

One example of the use of misaminoacylation to detect nascent protein isschematically represented in FIG. 4. A tRNA molecule is misaminoacylatedwith the marker which is highly fluorescent when excited with UV(ultraviolet) radiation. The misaminoacylated tRNA is then introducedinto a cell-free protein synthesis extract and the nascent proteinscontaining the marker analog produced. Proteins in the cell-free extractare separated by polyacrylamide gel electrophoresis (PAGE). Theresulting gel contains bands which correspond to all of the proteinspresent in the cell-free extract The nascent protein is identified uponV illumination of the gel by detection of fluorescence from the bandcorresponding to proteins containing marker. Detection can be throughvisible observation or by other conventional means of fluorescencedetection.

The misaminoacylated tRNA can be formed by natural aminoacylation usingcellular enzymes or misaminoacylation such as chemicalmisaminoacylation. One type of chemical misaminoacylation involvestruncation of the tRNA molecule to permit attachment of the marker ormarker precursor. For example, successive treatments with periodate pluslysine, pH 8.0, and alkaline phosphatase removes 3′-terminal residues ofany tRNA molecule generating tRNA-OH-3′ with a mononucleotide ordinucleotide deletion from the 3′-terminus (Neu and Heppel, J. Biol.Chem. 239:2927-34, 1964). Alternatively, tRNA molecules may begenetically manipulated to delete specific portions of the tRNA gene.The resulting gene is transcribed producing truncated tRNA molecules(Sampson and Uhlenbeck Proc. Natl. Acad. Sci. USA 85:1033-37, 1988).Next, a dinucleotide is chemically linked to a modified amino acid orother marker by, for example, acylation. Using this procedure, markerscan be synthesized and acylated to dinucleotides in high yield (Hudson,J. Org. Chem. 53:617-624, 1988; Happ et al., J. Org. Chem 52:5387-91,1987). These modified groups are bound together and linked via thedinucleotide to the truncated tRNA molecules in a process referred to asligase coupling (FIG. 3B).

A different bond is involved in misaminoacylation (FIG. 3B, link B) thanthe bond involved with activation of tRNA by aminoacyl tRNA synthetase(FIG. 3B, link A). As T4 RNA Ligase does not recognize the acylsubstituent, tRNA molecules can be readily misaminoacylated with fewchemical complications or side reactions (link B. FIG. 3B) (T. G.Heckler et al., Biochemistry 23:1468-73, 1984; and T. G. Heckler et al.,Tetrahedron 40:87-94, 1984). This process is insensitive to the natureof the attached amino acid and allows for misaminoacylation using avariety of non-native amino acids. In contrast, purely enzymaticaminoacylation (link A) is highly sensitive and specific for thestructures of substrate tRNA and amino acids.

Markers are basically molecules which will be recognized by the enzymesof the translation process and transferred from a charged tRNA into agrowing peptide chain To be useful, markers must also possess certainphysical and physio-chemical properties. Therefore, there are multiplecriteria:which can be used to identify a useful marker. First, a markermust be suitable for incorporation into a growing peptide chain. Thismay be determined by the presence of chemical groups which willparticipate in peptide bond formation. Second, markers should beattachable to a tRNA molecule. Attachment is a covalent interactionbetween the 3′-terminus of the tRNA molecule and the carbon group of themarker or a linking group attached to the marker and to a truncated tRNAmolecule. Linking groups may be nucleotides, short oligonucleotides orother similar molecules and are preferably dinucleotides and morepreferably the dinucleotide CA. Third, markers should have one or morephysical properties that facilitate detection and possibly isolation ofnascent proteins. Useful physical properties include a characteristicelectromagnetic spectral property such as emission or absorbance,magnetism, electron spin resonance, electrical capacitance, dielectricconstant or electrical conductivity.

Useful markers are native amino acids coupled with a detectable label,detectable non-native amino acids, detectable amino acid analogs anddetectable amino acid derivatives. Labels and other detectable moietiesmay be ferromagnetic, paramagnetic, diamagnetic, luminescentelectrochemiluminescent, fluorescent, phosphorescent or chromatic.Fluorescent moieties which are useful as markers include dansylfluorophores, coumarins and coumarin derivatives, fluorescent acridiniummoieties and benzopyrene based fluorophores. Preferably, the fluorescentmarker has a high quantum yield of fluorescence at a wavelengthdifferent from native amino acids. Upon excitation at a preselectedwavelength, the marker is detectable at low concentrations eithervisually or using conventional fluorescence detection methods.Electrochemiluminescent markers such as ruthenium chelates and itsderivatives or nitroxide amino acids and their derivatives are preferredwhen extreme sensitivity is desired (J. DiCésare et al., BioTechniques15:152-59, 1993). These markers are detectable at the fentomolar rangesand below.

In addition to fluorescent markers, a variety of other markerspossessing specific physical properties can be used to detect nascentprotein production. In general, these properties are based on theinteraction and response of the marker to electromagnetic fields andradiation and include absorption in the UV, visible and infrared regionsof the electromagnetic spectrum, presence of chromophores which areRaman active, and can be further enhanced by resonance Ramanspectroscopy, electron spin resonance activity and nuclear magneticresonances. These electromagnetic spectroscopic properties arepreferably not possessed by native amino acids or are readilydistinguishable from the properties of native amino acids. For example,the amino acid tryptophan absorbs near 290 nm, and has fluorescentemission near 340 nm when excited with light near 290 nm. Thus,tryptophan analogs with absorption and/or fluorescence properties thatare sufficiently different from tryptophan can be used to facilitatetheir detection in proteins.

Many different modified amino acids which can be used as markers arecommercially available (Sigma Chemical; St., Louis, Mo.; MolecularProbes; Eugene, Oreg.). One such marker is N_(ε)-dansyllysine created bythe misaminoacylation of a dansyl fluorophore to a tRNA molecule (FIG.5; scheme 1). The α-amino group of N_(ε)-dansyllysine is first blockedwith NVOC (ortho-nitro veratryl oxycarbonyl chloride) and the carboxylgroup activated with cyanomethyl ester. Misaminoacylation is performedas described. The misaminoacylated tRNA molecules are then introducedinto the protein synthesis system, whereupon the dansyllysine isincorporated directly into the newly synthesized proteins.

Another such marker is a fluorescent amino acid analog based on thehighly fluorescent molecule coumarin (FIG. 5; scheme 2). Thisfluorophore has a much higher fluorescence quantum yield than dansylchloride and can facilitate detection of much lower levels of nascentprotein. In addition, this coumarin derivative has a structure similarto the native amino acid tryptophan. These structural similarities areuseful where maintenance of the nascent proteins' native structure orfunction are important or desired. Coumarin is synthesized as depictedin FIG. 5 (scheme 2). Acetamidomalonate is alkylated with a slightexcess of 4-bromomethyl coumarin (Aldrich Chemicals; Milwaukee; Wis.) inthe presence of sodium ethoxide followed by acid hydrolysis. Thecorresponding amino acid as a hydrochloride salt that can be convertedto the free amino acid analog.

The coumarin derivative can be used most advantageously if itmisaminoacylates the tryptophan-tRNA, either enzymatically orchemically. When introduced in the form of the misaminoacylatedtryptophan-tRNA, the coumarin amino acid will be incorporated only intotryptophan positions. By controlling the concentration ofmisaminoacylated tRNAs or free coumarin derivatives in the cell-freesynthesis system, the number of coumarin amino acids incorporated intothe nascent protein can also be controlled. This procedure can beutilized to control the amount of most any markers in nascent proteins.

Markers can be chemically synthesized from a native amino acid and amolecule with marker properties which cannot normally function as anamino acid. For example a highly fluorescent molecule can be chemicallylinked to a native amino acid group. The chemical modification occurs onthe amino acid side-chain, leaving the carboxyl and aminofunctionalities free to participate in a polypeptide bond formation.Highly fluorescent dansyl chloride can be linked to the nucleophilicside chains of a variety of amino acids including lysine, arginine,tyrosine, cysteine, histidine, etc., mainly as a sulfonamide for aminogroups or sulfate bonds to yield fluorescent derivatives. Suchderivatization leaves the ability to form peptide bond intact, allowingthe normal incorporation of dansyllysine into a protein.

A marker can also be modified after the tRNA molecule is aminoacylatedor misaminoacylated using chemical reactions which specifically modifythe marker without significantly altering the functional activity of theaminoacylated tRNA. These types of post-aminoacylation modifications mayfacilitate detection, isolation or purification, and can sometimes beused where the modification allow the nascent protein to attain a nativeor more functional configuration.

Fluorescent and other markers which have detectable electromagneticspectral properties that can be detected by spectrometers anddistinguished from the electromagnetic spectral properties on nativeamino acids. Spectrometers which are most useful include fluorescence,Raman, absorption, electron spin resonance, visible, infrared andultraviolet spectrometers. Other markers, such as markers with distinctelectrical properties can be detected by an apparatus such as anammeter, voltmeter or other spectrometer. Physical properties of markerswhich relate to the distinctive interaction of the marker with anelectromagnetic field is readily detectable using instruments such asfluorescence, Raman, absorption, electron spin resonance spectrometers.Markers may also undergo a chemical, biochemical, electrochemical orphotochemical reaction such as a color change in response to externalforces or agents such as an electromagnetic field or reactant moleculeswhich allows its detection.

Normally detection first involves physical separation of the nascentproteins from other biomolecules present in the cellular or cell-freeprotein synthesis system. Protein separation can be performed using, forexample, gel electrophoresis or column chromatography and can be furtherfacilitated with affinity markers which uniquely bind acceptor groups.Detection of a marker containing a fluorophore by gel electrophoresiscan be accomplished using conventional fluorescence detection methods.

After protein synthesis in a cell-free system, the reaction mixture,which contains all of the biomolecules necessary for protein synthesisas well as nascent proteins, is loaded onto a gel which may be composedof polyacrylamide or agarose (R. C. Allen et al., Gel Electrophoresisand Isoelectric Focusing of Proteins, Walter de Gruyter, New York 1984).This mixture also contains the misaminoacylated tRNAs bearing the markeras well as uncharged tRNAs. Subsequent to loading the reaction mixture,a voltage is applied which spatially separates the proteins on the gelin the direction of the applied electric field. The proteins separateand appear as a set of discrete or overlapping bands which can bevisualized using a pre- or post-gel staining technique such as Coomasieblue staining. The migration of the protein band on the gel is afunction of the molecular weight of the protein with increasing distancefrom the loading position being a function of decreasing molecularweight. Bands on the gel which contain nascent proteins will exhibitfluorescence when excited at a suitable wavelength. These bands can bedetected visually, photographically or spectroscopically and, ifdesired, the nascent proteins purified from gel sections.

For example, if using dansyllysine as a marker, nascent proteins willfluoresce at 470 nm when excited by UV illumination. This fluorescencecan be detected visually by simply using a standard hand-held UVilluminator or a transilluminator. Approximately 10 nanograms (ng) ofthe protein bacteriorhodopsin is detectable using this method. Alsouseful are electronic imaging devices which can rapidly screen andidentify very low concentrations of markers.

The molecular weight and quantity of the nascent protein can bedetermined by comparison of its band-position on the gel with a set ofbands of proteins of predetermined molecular weight. For example, anascent protein of molecular weight 25,000 could be determined becauseof its relative position on the gel relative to a calibration gelcontaining the commercially available standard marker proteins of knownquantities and with known molecular weights (bovine serum albumin, 66kD; porcine heart fumarase, 48.5 kD; carbonic anhydrase, 29 kD,β-lactoglobulin, 18.4 kD; α-lactoglobulin, 142 kD; Sigma Chemical; St.Louis, Mo.).

Calibration proteins may also contain a similar markers for convenientdetection using the same method as the gel bearing the nascent protein.This can be accomplished in many cases by directly reacting thecalibration proteins with a molecule similar to the marker. For example,the calibration proteins can be modified with dansyl chloride so as toobtain their fluorescent derivatives (R. E. Stephens, Anal. Biochem. 65,369-79, 1975). These fluorescent proteins can be analyzed using PAGE.Combined detection of these fluorescent calibration proteins along withthat of nascent protein which contains fluorescent marker analog willaccurately determine both the molecular weight and quantity of thenascent protein synthesized. If necessary, the amounts of marker withineach calibration and nascent protein can be determined to provide anaccurate quantitation.

Other methods of protein separation are also useful for detection andsubsequent isolation and purification of nascent proteins containingmarkers. For example, proteins can be separated using capillaryelectrophoresis, isoelectric focusing, low pressure chromatography andhigh-performance or fast-pressure liquid chromatography (HPLC or FPLC).In these cases, the individual proteins are separated into fractionswhich can be individually analyzed by fluorescent detectors at theemission wavelengths of the markers. Alternatively, on-line fluorescencedetection can be used to detect nascent proteins as they emerge from thecolumn fractionation system. A graph of fluorescence as a function ofretention time provides information on both the quantity and purity ofnascent proteins produced.

Another embodiment of the invention is directed to a method forlabeling, detecting and, if desired, isolating and purifying nascentproteins, as described above, containing cleavable markers. Cleavablemarkers comprise a chemical structure which is sensitive to externaleffects such as physical or enzymatic treatments, chemical or thermaltreatments, electromagnetic radiation such as gamma rays, x-rays,ultraviolet light, visible light, infrared light, microwaves, radiowaves or electric fields. The marker is aminoacylated to tRNA moleculesas before using conventional technology or misaminoacylated and added toa translation system After incubation and production of nascentproteins, marker can be cleaved by the application of specifiedtreatments and nascent proteins detected. Alternatively, nascentproteins may also be detected and isolated by the presence or absence ofthe cleaved marker or the chemical moiety removed from the marker.

One example of a cleavable marker is a photocleavable marker such aschemical compounds which contain the 2-nitrobenzyl moiety (FIG. 6A).Upon illumination, these aromatic nitro compounds undergo an internaloxidation-reduction reaction (Pillai, Synthesis 1-26, 1980; Patchorniket al., J. Am. Chem. Soc. 92:6333-35, 1970). As a result, the nitrogroup is reduced to a nitroso group and an oxygen is inserted into thebenzylic carbon-hydrogen bond at the ortho position. The primaryphotochemical process is the intramolecular hydrogen abstraction by theexcited nitro group. This is followed by an electron-redistributionprocess to the aci-nitro form which rearranges to the nitroso product.Subsequent thermal reaction leads to the cleavage of substrate from thenitrobenzyl linkage (FIG. 6B). Examples of other cleavable markers areshown in FIG. 7.

It may sometimes be desirable to create a distance between the substrateand the cleavable moiety. To accomplish this, cleavable moieties may beseparated from substrates by cross-linker arms. Cross-linkers increasesubstrate access and stabilize the chemical structure, and can beconstructed using, for example, long alkyl chains or multiple repeatunits of caproyl moieties linked via amide linkages.

There are as many methods to synthesize cleavable markers as there aredifferent markers. One example for the synthesis of photocleavablebiotins based on nitrobenzyl alcohols involves four major steps.2-bromo-2′-nitroacetphenone, a precursor of the photoreactive moiety, isfirst converted into au α- or ω-amino acid like ε-aminocaprylic acid.The resulting acid and amino functionality of the photoreactive group iscoupled using dicyclohexyl carbodiimide (DCC). The benzoyl carbonylgroup of the resulting amide is reduced using sodium borohydride. Theresulting derivative of nitrobenzyl alcohol is derivatized to obtain thefinal component which is able to react with biomolecular substrates, forexample by the reaction with phosgene gas, to introduce thechloroformate functionality. The resulting compound is depicted in FIG.8A along with alternative derivatives of PCB. Possible linkages to aminoacids are depicted in FIG. 8B.

Cleavable markers can facilitate the isolation of nascent proteins. Forexample, one type of a cleavable marker is photocleavable biotin coupledto an amino acid. This marker can be incorporated into nascent proteinsand the proteins purified by the specific interaction of biotin withavidin or streptavidiz Upon isolation and subsequent purification, thebiotin is removed by application of electromagnetic radiation andnascent proteins utilized in useful applications without thecomplications of an attached biotin molecule. Other examples ofcleavable markers include photocleavable coumarin, photocleavabledansyt, photocleavable dinitrophenyl and photocleavable coumarin-biotin.Photocleavable markers are cleaved by electromagnetic radiation such asW light, peptidyl markers are cleaved by enzymatic treatments, andpyrenyl fluorophores linked by disulfide bonds are cleaved by exposureto certain chemical treatments such as thiol reagents.

Cleavage of photocleavable markers is dependent on the structure of thephotoreactive moiety and the wavelength of electromagnetic radiationused for illumination. Other wavelengths of electromagnetic radiationshould not damage the proteins or other chemical moieties. In the caseof unsubstituted 2-nitrobenzyl derivatives, the yield of photolysis andrecovery of the substrate are significantly decreased by the formationof side products which act as internal light filters and are capable toreact with amino groups of the substrate. Typical illumination timesvary from 1 to about 24 hours and yields are 1-95%. Radiation sourcesare placed within about 10 cm of the substrate proteins and set on lowpower so as to minimize side reactions, if any, which may occur in thenascent proteins. In the case of alpha-substituted 2-nitrobenzylderivatives (methyl, phenyl, etc.), a considerable increase in rate ofphoto-removal as well as yield of the released substrate are observed.The introduction of other electron donor groups into phenyl rings ofphotoreactive moieties increases the yield of substrate. The generalreaction for the photolysis of PCB is depicted in FIG. 9.

For enzymatic cleavage, markers introduced contain specific bonds whichare sensitive to unique enzymes of chemical substances. Introduction ofthe enzyme or chemical into the protein mixture cleaves the marker fromthe nascent protein. When the marker is a modified amino acid, this canresult in the production of native protein forms. Thermal treatments of,for example, heat sensitive chemical moieties operate in the samefashion Mild application of thermal energy, such as with microwaves orradiant heat, cleaves the sensitive marker from the protein withoutproducing any significant damage to the nascent proteins.

Another embodiment of the invention is directed to a method formonitoring the synthesis of nascent proteins in a cellular or acell-free protein synthesis system without separating the components ofthe system. These markers have the property that once incorporated intothe nascent protein they are distinguishable from markers free insolution or linked to a tRNA This type of marker, also called areporter, provides a means to detect and quantitate the synthesis ofnascent proteins directly in the cellular or cell-free translationsystem.

Reporters have the characteristic that once incorporated into thenascent protein by the protein synthesizing system, they undergo achange in at least one of their physical or physio-chemical properties.The resulting nascent protein can be uniquely detected inside thesynthesis system without the need to separate or partially purify theprotein synthesis system into its component parts. This type of markerprovides a convenient non-radioactive method to monitor the productionof nascent proteins without the necessity of first separating them frompre-existing proteins in the protein synthesis system. A reporter markerwould also provide a means to detect and distinguish between differentnascent proteins produced at different times during protein synthesis byaddition of markers whose properties are distinguishable from eachother, at different times during protein expression. This would providea means of studying differential gene expression.

One example of the utilization of reporters is schematically representedin FIG. 10. A tRNA molecule is misaminoacylated with a reporter (R)which has lower or no fluorescence at a particular wavelength formonitoring and excitation. The misaminoacylated. tRNA is then introducedinto a cellular or cell-free protein synthesis system and the nascentproteins containing the reporter analog are gradually produced. Uponincorporation of the reporter into the nascent protein (R*), it exhibitsan increased fluorescence at known wavelengths. The gradual productionof the nascent protein is monitored by detecting the increase offluorescence at that specific wavelength.

The chemical synthesis of a reporter can be based on the linkage of achemical moiety or a molecular component having reporter properties witha native amino acid residue. There are many fluorescent molecules whichare sensitive to their environment and undergo a change in thewavelength of emitted light and yield of fluorescence. When thesechemical moieties, coupled to amino acids, are incorporated into thesynthesized protein their environments are altered because of adifference between the bulk aqueous medium and the interior of a proteinwhich can causes reduced accessibility to water, exposure to chargedionic groups, reduced mobility, and altered dielectric constant of thesurrounding medium. Two such examples are shown in FIG. 11A.

One example of a reporter molecule is based on a fluorescent acridiniummoiety and has the unique property of altering its emission properties,depending upon polarity or viscosity of the microenvironment. It has ahigher quantum yield of fluorescence when subjected to hydrophobicenvironment and/or viscosity. Due to the hydrophobicity of the reporteritself, it is more likely to be associated with the hydrophobic core ofthe nascent protein after incorporation into the growing nascentpolypeptide. An increase in the fluorescence intensity is a directmeasure of protein synthesis activity of the translation system.Although, the environment of each reporter residue in the protein willbe different, and in some cases, the reporter may be present on thesurface of the protein and exposed to an aqueous medium, a net changeoccurs in the overall spectroscopic properties of the reportersincorporated into the protein relative to bulk aqueous medium. A changein the spectroscopic properties of only a subset of reporters in theprotein will be: sufficient to detect the synthesis of proteins thatincorporate such reporters.

An alternative approach is to utilize a reporter which alters itsfluorescent properties upon formation of a peptide bond and notnecessarily in response to changes in its environment Changes in thereporter's fluorescence as it partitions between different environmentsin the cell-free extract does not produce a large signal change comparedto changes in fluorescence upon incorporation of the reporter into thenascent protein.

A second example of a reporter is a marker based on coumarin such as6,7-(4′, 5′-prolino)coumarin. This compound can be chemicallysynthesized by coupling a fluorophore like coumarin with an amino-acidstructural element in such a way that the fluorophore would alter itsemission or absorption properties after forming a peptide linkage (FIG.11B). For example, a proline ring containing secondary amino functionswill participate in peptide bond formation similar to a normal primaryamino group. Changes in fluorescence occur due to the co-planarity ofthe newly formed peptide group in relation to the existing fluorophore.This increases conjugation/delocalization due to the π-electrons ofnitrogen-lone pair and carbonyl-group in the peptide bond. Synthesis ofsuch compounds is based on on coumarin synthesis using ethylacetoacetate(FIG. 11C).

Reporters are not limited to those non-native amino acids which changetheir fluorescence properties when incorporated into a protein. Thesecan also be synthesized from molecules that undergo a change in otherelectromagnetic or spectroscopic properties including changes inspecific absorption bands in the UV, visible and infrared regions of theelectromagnetic spectrum, chromophores which are Raman active and can beenhanced by resonance Raman spectroscopy, electron spin resonanceactivity and nuclear magnetic resonances. In general, a reporter can beformed from molecular components which undergo a change in theirinteraction and response to electromagnetic fields and radiation afterincorporation into the nascent protein.

Reporters may also undergo a change in at least one of their physical orphysiochemical properties due to their interaction with other markerswhich are incorporated into the same nascent protein. The interaction oftwo different markers with each other causes them to become specificallydetectable. One type of interaction would be a resonant energy transferwhich occurs when two markers are within a distance of between about 1angstrom (Å) to about 50 Å, and preferably less than about 10 Å. In thiscase, excitation of one marker with electromagnetic radiation causes thesecond marker to emit electromagnetic radiation of a differentwavelength which is detectable. A second type of interaction would bebased on electron transfer between the two different markers which canonly occur when the markers are less than about 5 Å. A third interactionwould be a photochemical reaction between two markers which produces anew species that has detectable properties such as fluorescence.Although these markers are also present on the misaminoacylated tRNAsused for their incorporation into nascent proteins, the interaction ofthe markers occurs primarily when they are incorporated into protein dueto their close proximity. In certain cases, the proximity of two markersin the protein can also be enhanced by choosing tRNA species that willinsert markers into positions that are close to each other in either theprimary, secondary or tertiary structure of the protein. For example, atyrosine-tRNA and a tryptophan-tRNA could be used to enhance theprobability for two different markers to be near each other in a proteinsequence which contains the unique neighboring pair tyrosine-tryptophan.

As stated above, a principal advantage of using reporters is the abilityto monitor the synthesis of proteins in cellular or a cell-freetranslation systems directly without further purification or isolationsteps. Reporter markers may also be utilized in conjunction withcleavable markers that can remove the reporter property at will. Suchtechniques are not available using radioactive amino acids which requirean isolation step to distinguish the incorporated marker from theunincorporated marker. With in vitro translation systems, this providesa means to determine the rate of synthesis of proteins and to optimizesynthesis by altering the conditions of the reaction. For example, an invitro translation system could be optimized for protein production bymonitoring the rate of production of a specific calibration protein. Italso provides a dependable and accurate method for studying generegulation in a cellular or cell-free systems.

Another embodiment of the invention is: directed to the use of markersthat facilitate the detection or separation of nascent proteins producedin a cellular or cell-free protein synthesis system. Such markers aretermed affinity markers and have the property that they selectivelyinteract with molecules and/or materials containing acceptor groups. Theaffinity markers are linked by aminoacylation to tRNA molecules in anidentical manner as other markers of non-native amino acid analogs andderivatives and reporter-type markers as described. These affinitymarkers are incorporated into nascent proteins once the misaminoacylatedtRNAs are introduced into a translation system.

An affinity marker facilities the separation of nascent proteins becauseof its selective interaction with other molecules which may bebiological or non-biological in origin through a coupling agent. Forexample, the specific molecule to which the affinity marker interacts,referred to as the acceptor molecule, could be a small organic moleculeor chemical group such as a sulphydryl group (—SH) or a largebiomolecule such as an antibody. The binding is normally chemical innature and may involve the formation of covalent or non-covalent bondsor interactions such as ionic or hydrogen bonding. The binding moleculeor moiety might be free in solution or itself bound to a surface, apolymer matrix, or a reside on the surface of a substrate. Theinteraction may also be triggered by an external agent such as Light,temperature, pressure or the addition of a chemical or biologicalmolecule which acts as a catalyst.

The detection and/or separation of the nascent protein and otherpreexisting proteins in the reaction mixture occurs because of theinteraction, normally a type of binding, between the affinity marker andthe acceptor molecule. Although, in some cases some incorporatedaffinity marker will be buried inside the interior of the nascentprotein, the interaction between the affinity marker and the acceptormolecule will still occur as long as some affinity markers are exposedon the surface of the nascent protein. This is not normally a problembecause the affinity marker is distributed over several locations in theprotein sequence.

Affinity markers include native amino acids, non-native amino acids,amino acid derivatives or amino acid analogs in which a coupling agentis attached or incorporated. Attachment of the coupling agent to, forexample, a non-native amino acid may occur through covalentinteractions, although non-covalent interactions such as hydrophilic orhydrophobic interactions, hydrogen bonds, electrostatic interactions ora combination of these forces are also possible. Examples of usefulcoupling agents include molecules such as haptens, immunogenicmolecules, biotin and biotin derivatives, and fragments and combinationsof these molecules. Coupling agents enable the selective binding orattachment of newly formed nascent proteins to facilitate theirdetection or isolation. Coupling agents may contain antigenic sites fora specific antibody, or comprise molecules such as biotin which is knownto have strong binding to acceptor groups such as streptavidin. Forexample, biotin may be covalently linked to an amino acid which isincorporated into a protein chain. The presence of the biotin willselectively bind only nascent proteins which incorporated such markersto avidin molecules coated onto a surface. Suitable surfaces includeresins for chromatographic separation plastics such as tissue culturesurfaces for binding plates, microtiter dishes and beads, ceramics andglasses, particles including magnetic particles, polymers and othermatrices. The treated surface is washed with, for example, phosphatebuffered saline (PBS), to remove non-nascent proteins and othertranslation reagents and the nascent proteins isolated. In some casethese materials may be part of biomolecular sensing devices such asoptical fibers, chemfets, and plasmon detectors.

One example of an affinity marker is dansyllysine (FIG. 5). Antibodieswhich interact with the dansyl ring are commercially available (SigmaChemical; St. Louis, Mo.) or can be prepared using known protocols suchas described in Antibodies: A Laboratory Manual (E. Harlow and D. Lane,editors, Cold Spring Harbor Laboratory Press, 1988) which is herebyspecifically incorporated by reference. Many conventional techniquesexist which would enable proteins containing the dansyl moiety to beseparated from other proteins on the basis of a specific antibody-dansylinteraction. For example, the antibody could be immobilized onto thepacking material of a chromatographic column. This method, known asaffinity column chromatography, accomplishes protein separation bycausing the target protein to be retained on the column due to itsinteraction with the immobilized antibody, while other proteins passthrough the column. The target protein is then released by disruptingthe antibody-antigen interaction. Specific chromatographic columnmaterials such as ion-exchange or affinity Sepharose, Sephacryl,Sephadex and other chromatography resins are commercially available(Sigma Chemical; St. Louis, Mo.; Pharmacia Biotech; Piscataway, N.J.).

Separation can also be performed through an antibody-dansyl interactionusing other biochemical separation methods such as immunoprecipitationand immobilization of the antibodies on filters or other surfaces suchas beads, plates or resins. For example, protein could be isolated bycoating magnetic beads with a protein-specific antibody. Beads areseparated from the extract using magnetic fields. A specific advantageof using dansyllysine as an affinity marker is that once a protein isseparated it can also be conveniently detected because of itsfluorescent properties.

In addition to antibodies, other biological molecules exist whichexhibit equally strong interaction with target molecules or chemicalmoieties. An example is the interaction of biotin and avidin. In thiscase, an affinity analog which contains the biotin moiety would beincorporated into the protein using the methods which are part 0f thepresent invention. Biotin-lysine amino acid analogs are commerciallyavailable (Molecular Probes; Eugene, Oreg.).

Affinity markers can also comprise cleavable markers incorporating acoupling agent. This property is important in cases where removal of thecoupled agent is required to preserve the native structure and functionof the protein and to release nascent protein from acceptor groups. Insome cases, cleavage and removal of the coupling agent results inproduction of a native amino acid. One such example is photocleavablebiotin coupled to an amino acid.

Photocleavable biotin contains a photoreactive moiety which comprises aphenyl ring derivatized with functionalities represented in FIG. 12 byX, Y and Z. X allows linkage of PCB to the bimolecular substrate throughthe reactive group X′. Examples of X′ include Cl,O—N-hydroxysuccinildyl, OCH₂CN, OPhF₅, OPhCl₅, N₃. Y represents asubstitution pattern of a phenyl ring containing one or moresubstitutions such as nitro or alkoxyl. The functionality Z represents agroup that allows linkage of the cross-linker moiety to thephotoreactive moiety. The photoreactive moiety has the property thatupon illumination, it undergoes a photoreaction that results in cleavageof the PCB molecule from the substrate.

A lysine-tRNA is misaminoacylated with photocleavable biotin-lysine, orchemically modified to attach a photocleavable biotin amino acid. Themisaminoacylated tRNA is introduced into a cell-free proteinsynthesizing system and nascent proteins produced. The nascent proteinscan be separated from other components of the system bystreptavidin-coated magnetic beads using conventional methods which relyon the interaction of beads with a magnetic field. Nascent proteins arereleased then from beads by irradiation with UV light of approximately280 nm wavelength.

Many devices designed to detect proteins are based on the interaction ofa target protein with specific immobilized acceptor molecule. Suchdevices can also be used to detect nascent proteins once they containaffinity markers such as biodetectors based on sensing changes insurface plasmons, light scattering and electronic properties ofmaterials that are altered due to the interaction of the target moleculewith the immobilized acceptor group.

Nascent proteins, including those which do not contain affinity-typemarkers, may be isolated by more conventional isolation techniques. Someof the more useful isolation techniques which can be applied or combinedto isolate and purify nascent proteins include chemical extraction, suchas phenol or chloroform extract, dialysis, precipitation such asammonium sulfate cuts, electrophoresis, and chromatographic techniques.Chemical isolation techniques generally do not provide specificisolation of individual proteins, but are useful for removal of bulkquantities of non-proteinaceous material. Electrophoretic separationinvolves placing the translation mixture containing nascent proteinsinto wells of a gel which may be a denaturing or non-denaturingpolyacrylamide or agarose gel. Direct or pulsed current is applied tothe gel and the various components of the system separate according tomolecular size, configuration, charge or a combination of their physicalproperties. Once distinguished on the gel, the portion containing theisolated proteins removed and the nascent proteins purified from thegel. Methods for the purification of protein from acrylamide and agarosegels are known and commercially available.

Chromatographic techniques which are useful for the isolation andpurification of proteins include gel filtration, fast-pressure orhigh-pressure liquid chromatography, reverse-phase chromatography,affinity chromatography and ion exchange chromatography. Thesetechniques are very useful for isolation and purification of proteinsspecies containing selected markers.

Another embodiment of the invention is directed to the incorporation ofnon-native amino acids or amino acid derivatives with marker or affinityproperties at the amino-terminal residue of a nascent protein (FIG. 13).This can be accomplished by using the side chain of an amino acid or byderivatizing the terminal amino group of an amino acid. In either casethe resulting molecule is termed an amino acid derivative.

The amino-terminal residue of a protein is free and its derivatizationwould not interfere with formation of the nascent polypeptide. Thenon-native amino acid or amino acid derivative is then used tomisaminoacylate an initiator tRNA which only recognizes the first AUGcodon signaling the initiation of protein synthesis. After introductionof this misaminoacylated initiator tRNA into a protein synthesis systemmarker is incorporated only at the amino terminal of the nascentprotein. The ability to incorporate at the N-terminal residue can beimportant as these nascent molecules are most likely to fold into nativeconformation. This can be useful in studies where detection or isolationof functional nascent proteins is desired.

It may often be advantageous to incorporate more than one marker into asingle species of protein. This can be accomplished by using a singletRNA species such as a lysine tRNA misaminoacylated with both a markersuch as dansyllysine and a coupling agent such as biotin-lysine.Alternatively, different tRNAs which are each misaminoacylated withdifferent markers can also be utilized. For example, the coumarinderivative could be used to misaminoacylate a tryptophan tRNA and adansyl-lysine used to misaminoacylate a lysine tRNA.

One use of multiple misaminoacylated tRNAs is to study the expression ofproteins under the control of different genetic elements such asrepressors or activators, or promoters or operators. For example, thesynthesis of proteins at two different times in response to an internalor external agent could be distinguished by introducing misaminoacylatedtRNAs at different times into the cellular or cell-free proteinsynthesis system. A tRNA^(tyr) might be charged with marker A and atRNA^(tyr) charged with marker B, yielding A-tRNA^(tyr) andB-tRNA^(tyr), respectively. In this case, protein one under the controlof one promoter can be labeled by adding the A-tRNA^(tyr) to thereaction system. If a second misaminoacylated tRNA, B-tRNA^(tyr) is thenadded and a second promoter for protein two activated, the nascentprotein produced will contain both label A and B. Additional markerscould also be added using additional tRNA molecules to further study theexpression of additional proteins. The detection and analysis ofmultiply labeled nascent proteins can be facilitated by using themulti-colored electrophoresis pattern reading system, described in U.S.Pat. No. 5,190,632, which is specifically incorporated by reference, orother multi-label reading systems such as those described in U.S. Pat.Nos. 5,069,769 and 5,137,609, which are both hereby specificallyincorporated by reference.

A second use of multiple misaminoacylated tRNAs is in the combinedisolation and detection of nascent proteins. For example, biotin-lysinemarker could be used to misaminoacylate one tRNA and a coumarin markerused to misaminoacylate a different tRNA. Magnetic particles coated withstreptavidin which binds the incorporated lysine-biotin would be used toisolate nascent proteins from the reaction mixture and the coumarinmarker used for detection and quantitation.

A schematic diagram of the basics of the above methods is shown in FIG.14. In a first step, the marker selected (M), which may have reporter(R) or affinity (A) properties, is chemically or enzymaticallymisaminoacylated to a single tRNA species or a mixture of differenttRNAs. Prior to protein synthesis, a predetermined amount of themisaminoacylated tRNA, charged with the fluorescent marker is mixed withthe cell-free protein synthesis reaction system at concentrationssufficient to allow the misaminoacylated tRNA to compete effectivelywith the corresponding tRNA. After an incubation of about 1-3 hours, thereaction mixture is analyzed using conventional polyacrylamide oragarose gel electrophoresis. After electrophoresis, the gel isilluminated by UV radiation. Bands due to the nascent protein exhibitdistinct fluorescence and can be easily and rapidly distinguished,either visually or photographically, from non-fluorescent bands ofpreexisting proteins. Nascent proteins can be isolated by excising thefluorescent band and electroeluting the protein from the extracted gelpieces. The quantities and molecular weights of the nascent proteins canbe determined by comparison of its fluorescence with the fluorescenceproduced by a set of proteins with known molecular weights and knownquantities. The results of the assay can be recorded and stored forfurther analysis using standard electronic imaging and photographic orspectroscopic methods.

Another embodiment of the invention is directed to a compositioncomprising nascent proteins isolated or purified by conventional methodsafter translation in the presence of markers. Compositions can beutilized in manufacturing for the preparation of reagents such ascoatings for tissue culture products and in the pharmaceutical industry.

Incorporation of markers into nascent proteins utilized in manufacturingfacilitates analysis of the final manufactured product or process bydetection of marker. For example, nascent proteins produced may be usedas coatings for tissue culture products. The reproducibility of aparticular coating process could be accurately determined by detectingvariations of marker emissions over the surface of the coated product.In addition, non-toxic markers incorporated into proteins encompassedwithin a pharmaceutical preparation such as a hormone, steroid, immuneproduct or cytokine can be utilized to facilitate safe and economicalisolation of that protein preparation. Such products could be useddirectly without the need for removal of marker. When very lowconcentrations of marker are preferred, limiting amounts of markedproteins could be used to follow a protein through a purificationprocedure. Such proteins can be efficiently purified and the purity ofthe resulting composition accurately determined. In addition, thepresence of markers may facilitate study and analysis of pharmaceuticalcompositions in testing. For example, markers can be utilized todetermine serum half-life, optimum serum levels and the presence orabsence of break-down products of the composition in a patient.

Alternatively, nascent proteins may contain specific markers which serveas therapeutically useful compounds such as toxic substances. Theseproteins are administered to a patient and the therapeutic moietyreleased after proteins have identified and possibly bound to theirrespective targets. Release may be electrical stimulation, photochemicalcleavage or other means whereby the moiety is specifically deposited inthe area targeted by the nascent proteins. In addition, moieties such asmodified toxins may be utilized which become toxic only after releasefrom nascent proteins. Nascent protein may also serve as apharmaceutical carrier which bestows the incorporated marker with activetherapeutic function or prevents marker from breaking down in the bodyprior to its therapeutic or imaging action.

The incorporation of cleavable markers in nascent proteins furtherprovides a means for removal of the non-native portion of the marker tofacilitate isolation of the protein in a completely native form. Forexample, a cleavable affinity marker such as photocleavable biotinintroduced into a nascent protein facilitates economical isolation ofthe protein and allows for the removal of the marker for further use asa pharmaceutical composition.

Pharmaceutical compositions of proteins prepared by translation in thepresence of markers may further comprise a pharmaceutically acceptablecarrier such as, for example, water, oils, lipids, polysaccharides,glycerols, collagens or combinations of these carriers. Usefulimmunological compositions include immunologically active compositions,such as a vaccine, and pharmaceutically active compositions, such as atherapeutic or prophylactic drug which can be used for the treatment ofa disease or disorder in a human.

Another embodiment of the invention is directed to diagnostic kits oraids containing, preferably, a cell-free translation containing specificmisaminoacylated tRNAs which incorporate markers into nascent proteinscoded for by mRNA or genes, requiring coupled transcription-translationsystems, and are only detectably present in diseased biological samples.Such kits may be useful as a rapid means to screen humans or otheranimals for the presence of certain diseases or disorders. Diseaseswhich may be detected include infections, neoplasias and geneticdisorders. Biological samples most easily tested include samples ofblood, serum, tissue, urine or stool, nasal cells or spinal fluid. Inone example, misaminoacylate fmet-tRNAs could be used as a means todetect the presence of bacteria in biological samples containingprokaryotic cells. Kits would contain translation reagents necessary tosynthesize protein plus tRNA molecules charged with detectablenon-radioactive markers. The addition of a biological sample containingthe bacteria-specific genes would supply the nucleic acid needed fortranslation. Bacteria from these samples would be selectively lysedusing a bacteria directed toxin such as Colicin E1 or some otherbacteria-specific permeabilizing agent. Specific genes from bacterialDNA could also be amplified using specific oligonucleotide primers inconjunction with polymerase chain reaction (PCR), as described in U.S.Pat. No. 4,683,195, which is hereby specifically incorporated byreference. Nascent proteins containing marker would necessarily havebeen produced from bacteria. Utilizing additional markers or additionaltypes of detection kits, the specific bacterial infection may beidentified.

Kits may also be used to detect specific diseases such as familialadenomatous polyposis. In about 30 to 60% of cases of familialadenomatous polyposis, the diseased tissues also contain chainterminated or truncated transcripts of the APC gene (S. M. Powell etal., N. Engl. J. Med. 329:1982-87, 1993). Chain termination occurs whenframeshift cause a stop codon such as UAG, UAA or UGA to appear in thereading frame which terminates translation Using misaminoacylated tRNAswhich code for suppressor tRNAs, such transcripts can be rapidly anddirectly detected in inexpensive kits. These kits would contain atranslation system, charged suppressor tRNAs containing detectablemarkers, for example photocleavable coumarin-biotin, and appropriatebuffers and reagents. A biological sample, such as diseased cells,tissue or isolated DNA or mRNA, is added to the system, the system isincubated and the products analyzed. Analysis and, if desired, isolationis facilitated by a marker such as photocleavable coumarin-biotin whichcan be specifically detected using streptavidin coupled to magneticbeads. Such kits would provide a rapid, sensitive and selectivenon-radioactive diagnostic assay for the presence or absence of thedisease.

A variety of proteins and protein derivatives useful in the treatment ofhuman diseases can be produced in a cell-free system This includestoxins which are difficult to grow in cellular systems because of thetoxicity to the cell, proteins involved in gene regulation,transcription, translation and cell-division. Examples include cis-A andrel which are difficult to express and produce in cellular systems dueto interference with the native mechanisms of the cell, and proteinsmodified with non-native amino acids for labeling of or localization inspecific human cells, or, for interaction with other biomolecules of acell. One example is the protein coded for the gene MTS1 which may bemissing or defective in more that 50% of all human cancer cells. Thesenascent proteins can be conveniently isolated from the cell-freesynthesis system by incorporation of detectable markers. For example, amethionine initiator tRNA can be misaminoacylated with a photocleavablebiotin amino acid or photocleavable coumarin-biotin amino acid which isintroduced into the cell-free synthesis system along with the plasmidscontaining the gene that codes for the desired protein product Thenascent protein is isolated from the cell-free reaction mixture by theintroduction of streptavidin coated magnetic beads. Irradiation withlight at between about 250 nm to about 350 nm, preferably between about280 nm to about 320 nm, and more preferably at about 300 nm, causes thecleavage of the bonds between photocleavable biotin and the coumarinamino acid.

Nascent proteins with defined therapeutic properties can be introducedto a patient such as a human as a preparation combined with apharmaceutically acceptable carrier. In some cases, marker may serve tocouple the nascent protein to a pharmaceutically acceptable carrier. Forexample, streptavidin immobilized on a biodegradable magnetic beadcarrier (K. J. Vidder et al, Proc. Soc. Exp. Biol. & Med. 58:14146,1978) such as the MicroImageETM beads offered commercially by theOmniQuest corporation can be used to bind a nascent proteinincorporating the marker photocleavable biotin. In the case of abiodegradable magnetic beads, composed of albumin the nascent protein isisolated from the reaction mixture as described above using such beadcoated with streptavidin. It is then introduced intra-arterially andcaused to concentrate at a tumor site by application of an externalmagnetic field with an external magnet or internal application of amagnetic field through surgical implantation of a small magnet. Once themagnetic bead carrier is concentrated, the therapeutic or marker proteinis released by the action of light which cleaves the photocleavablebiotin that binds the nascent protein to the magnetic beads oralternatively through degradation of the bead which may be enhancedthrough other mechanisms.

A nascent protein may contain a non-radioactive marker which is used todetermine the site of a tumor, pathogen or other abnormal tissues in thebody. One example of a marker protein would be a nascent protein thatincludes a non-radioactive marker which acts as an MRI image enhancer. Asecond would be a nascent protein which contains a fluorescent markersuch as coumarin or photocleavable coumarin. The nascent protein may bechosen to have a high affinity to specific:antigens that reside on thetumor cells, pathogenic virus or bacteria. The non-radioactive markermay also be used to monitor the half-life of the nascent protein. Forexample, a specific fluorescence signal from a reporter marker may bemonitored to determine the rate of proteolytic degradation of aparticular protein in the body.

Nascent proteins containing markers are also useful to enhance orreplace presently available protein products. One example involvesbacteriorhodopsin, which has photochemical properties including a rapidchange in its visible absorption spectrum and has been utilized in avariety of opto-electronic devices including spatial light modulators,real-time holographic interferometers, and photodetectors (D. Oesterheltet al., Quart. Rev. Biophys. 4:425-78, 1991). The incorporation offluorescent markers enhance the usefulness of bacteriorhodopsin in thesedevices by providing a means of determining the state ofbacteriorhodopsin through fluorescent emission. Further,bacteriorhodopsin can also be created with a photocleavable biotinmoiety. These markers are incorporated into proteins bymisaminoacylation. After isolation, the modified bacteriorhodopsin canbe reconstituted in halobacterial lipids (S. Sonar et al, Biochem. 32:13777-81, 1993) and incorporated into thin films by a process forproducing a molecular oriented film such as described in U.S. Pat. No.4,241,0050, which is hereby specifically incorporated by reference.These thin films can be utilized in a variety of opto-electronic devicessuch as in a spatial light modulator.

In a second example, PCB modified bacteriorhodopsin can be reconstitutedinto two-dimensional self-assembling arrays which are used as a templatefor producing a patterned overlayer such as described in U.S. Pat. Nos.4,728,591 and 4,802,951, which are hereby specifically incorporated byreference. This overlayer could consist of enzymes or other biomoleculeswhich selectively interact with the affinity marker and assemble into apattern that replicates the bacteriorbodopsin template pattern.Additional patterning could be accomplished by selectively irradiatingspecific areas with light causing release of the overlying enzymes or byusing a near field scanning optical microscope. Patterning of enzymes atthe nanometer level can be used in the production of molecular devices.

The following examples illustrate embodiments of the invention, butshould not be viewed as limiting the scope of the invention

EXAMPLES Example 1 Preparation of Markers.

Synthesis of Coumarin Amino Acid: 4-(Bromomethyl)-7-methoxy coumarin(FIG. 15, compound 1; 6.18 mmole) and diethylacetamidomalonate (FIG. 15,compound 2; 6.18 mmole) were added to a solution of sodium ethoxide inabsolute ethanol and the mixture refluxed for 4 hours. The intermediateobtained (FIG. 15, compound 3) after neutralization of the reactionmixture and chloroform extraction was further purified bycrystallization from methanolic solution. This intermediate wasdissolved in a mixture of acetone and HCl (1:1) and refluxed for onehour. The reaction mixture was evaporated to dryness, and the amino acidhydrochloride precipitated using acetone. This hydrochloride wasconverted to free amino acid (FIG. 15, compound 4) by dissolving in 50%ethanol and adding pyridine to pH 4-5. The proton (¹H) NMR spectrum ofthe free amino acid was as follows: (m.p. 274-276° C., decomp.) —OCH₃ (δ3.85 s, 3H), —CH₂— (δ 3.5 d, 2H), α-CH— (δ 2.9 t, 1H), CH—CO (δ 6.25 s,1H), ring H (δ 7.05 s, 1H), (δ 7.8 d, 2H).

Synthesis of Fmoc derivative of coumarin: Coumarin amino acid (1.14mmol) was reacted with Fluorenylmethyloxycarbonyl N-hydroxysuccinildylester (Fmoc-NHS ester) 1.08 mmol) in the presence of 1.14 mmol oftriethylamine for 30 minutes at room temperature. The reaction mixturewas acidified and the precipitate washed with 1 N HCl and dried. The NMRspectrum of the free amino acid was as follows: (MP 223-225° C.) —OCH₃(δ 3.85 s, 3H), —CH₂Br (δ 3.5 broad singlet, 2H), α-CH— (δ 3.0 t, 1H),CH—CO (δ 6.22 m, 1H), ring H (δ 7.05 s, 1H), (δ 7.8 d, 2H), fluorene HCH₂—CH (δ 4.2 m, 2H), CH₂—CH (δ 4.25 m, 1H), aromatic regions showedcharacteristic multiplets.

Synthesis of PCB: Photocleavable biotin was synthesized as describedbelow. 2-bromo, 2′-nitroacetophenone (FIG. 15, compound 5) was convertedfirst into its hexamethyltetraamommonium salt which was decomposed toobtain 2-amino, 2′-nitroacetophenone (FIG. 15, compound 6). BiotinN-hydroxysuccinimidyl ester (FIG. 15, compound 7; Sigma Chemical; StLouis, Mo.) was reacted with a 6-aminocaproic acid (FIG. 15, compound 8)to obtain the corresponding acid (FIG. 15, compound 9). This acid wascoupled with the 2-amino, 2′nitroacetophenone using DCC to obtain theketone (FIG. 15, compound 10). The ketone was reduced using sodiumborohydride to obtain the alcohol (FIG. 15, compound 11) which wasfurther converted into its chloroformate derivative (FIG. 15, compound12). The proton NMR spectrum of the derivative (compound 12) was asfollows: (δ 13 m, 3H), (δ 1.4 m, 2H), (δ 1.5 m, 5H) (δ 1.62 m, 1H), (δ2.1 t, 2H) (δ 2.4 t, 2H), (δ 2.6 d, 1H), (δ 2.8 m, 1H), (δ 3.0 t, 1H),(δ 3.1 m 1H), (δ 4.15 qt, 1H) (δ 4.42 qt, 1H), (δ 5.8 t, 1H), (δ 6.25 s,1H), (δ 6.45 s, 1H), (δ 7.5 t, 1H), (δ 7.75 m, 4H), (δ 7.9 d, 1H).

Example 2 Misaminoacylation of tRNA.

The general strategy used for generating misaminoacylated tRNA is shownin FIG. 16 and involved truncation of tRNA molecules, dinucleotidesynthesis (FIG. 17), aminoacylation of the dinucleotide (FIG. 18) andligase mediated coupling.

a) Truncated tRNA molecules were generated by periodate degradation inthe presence of lysine and alkaline phosphatase basically as describedby Neu and Heppel (J. Biol. Chem. 239:2927-34, 1964). Briefly, 4 mmolesof uncharged E. coli tRNA^(LYS) molecules (Sigma Chemical; St, Louis,Mo.) were truncated with two successive treatments of 50 mM sodiummetaperiodate and 0.5 M lysine, pH 9.0, at 60° C. for 30 minutes in atotal volume of 50 μl. Reaction conditions were always above 50° C. andutilized a 10-fold excess of metaperiodate. Excess periodate wasdestroyed treatment with 5 μl of 1M glycerol. The pH of the solution wasadjusted to 8.5 by adding 15 μl of Tris-HCl to a final concentration of0.1 M. The reaction volume was increased to 150 μl by adding 100 μl ofwater. Alkaline phosphatase (15 μl, 30 units) was added and the reactionmixture incubated again at 60° C. for two hours. Incubation was followedby ethanol precipitation of total tRNA, ethanol washing, drying thepellet and dissolving the pellet in 20 μl water. This process wasrepeated twice to obtain the truncated tRNA.

b) Dinucleotide synthesis was carried out basically as performed byHudson (J. Org. Chem. 53:617-24, .1988), and can be described as a threestep process, deoxycytidine protection, adenosine protection anddinucleotide synthesis.

Deoxycytidine protection: All reaction were conducted at roomtemperature unless otherwise indicated. First, the 5′ and 3′ hydroxylgroups of deoxycytidine were protected by reacting with 4.1 equivalentsof trimethylsilyl chloride for 2 hours with constant stirring. Exocyclicamine function was protected by reacting it with 1.1 equivalents ofFmoc-Cl for 3 hours. Deprotection of the 5′ and 3′ hydroxyl wasaccomplished by the addition of 0.05 equivalents of KF and incubationfor 30 minutes. The resulting product (FIG. 17, compound 19) wasproduced at an 87% yield. Phosphate groups were added by incubating thiscompound with 1 equivalent of bis-(2-chlorophenyl)phosphorochloridateand incubating the mixture for 2 hours at 0° C. The yield in this casewas 25-30%.

Adenosine protection: Trimethylsilyl chloride (4.1 equivalents) wasadded to adenosine residue and incubated for 2 hours, after which, 1.1equivalents of Fmoc-Cl introduced and incubation continued for 3 hours.The TMS groups were deprotected with 0.5 equivalents of fluoride ions asdescribed above. The Fmoc protected adenosine (compound 22) was obtainedin a 56% yield. To further protect the 2′-hydroxyl, compound 22 wasreacted with 1.1 equivalents of tetraisopropyl disiloxyl chloride(TIPDSCl₂) for 3 hours which produces compound 23 at a 68-70% yield. Thecompound was converted to compound 24 by incubation with 20 equivalentsof dihydropyran and 0.33 equivalents of p-toluenesulfonic acid indioxane for about 4-5 hours. This compound was directly convertedwithout isolation into compound 25 (FIG. 17) by the addition of 8equivalents of tetrabutyl ammonium fluoride in a mixture oftetrahydro-furan, pyridine and water.

Dinucleotide synthesis: The protected deoxycytidine, compound 20, andthe protected adenosine, compound 25 (FIG. 17), were coupled by theaddition of 1.1 equivalents of 2-chlorophenyl bis-(1-hydroxybenzotriazolyl) phosphate in tetrahydrofuran with constant stirring for30 minutes. This was followed by the addition of 13 equivalents ofprotected adenosine, compound 25, in the presence of N-methylimidazolefor 30 minutes. The coupling yield was about 70% and the proton NMRspectrum of the coupled product, compound 26 (FIG. 17), was as follows:(δ 8.76 m, 2H), (δ 8.0 m, 3H), (δ 7.8 m, 3H) (δ 7.6 m, 4H), (δ 7.5 m,3H), (δ 7.4 m, 18H), (δ 7.0 m, 2H), (δ 4.85 m, 14H), (δ 4.25 m, 1H); (δ3.6 m, 2H), (δ 3.2 m, 2H), (δ 2.9 m, 3H), (δ 2.6 m, 1H), (δ 2.0-1.2 m,7H).

c) Aminoacylation of the dinucleotide was accomplished by linking theprotected marker amino acid, Fmoc-coumarin, to the dinucleotide with anester linkage.

First, the protected amino acid was activated with 6 equivalents ofbenzotriazol-1-yl-oxy tris-(dimethylamino) phosphonium hexafluorophosphate and 60 equivalents of 1-hydroxybenzotriazole intetrahydrofuran. The mixture was incubated for 20 minutes withcontinuous stirring. This was followed with the addition of 1 equivalentof dinucleotide in 3 equivalents N-methylimidazole, and the reactioncontinued at room temperature for 2 hours. Deprotection was carried outby the addition of tetramethyl guanidine and 4-nitrobenzaldoxime, andcontinuous stirring for another 3 hours. The reaction was completed bythe addition of acetic acid and incubation, again with continuousstirring for 30 minutes at 0° C. which produced the aminoacylateddinucleotide (FIG. 18).

d) Ligation of the tRNA to the aminoacylated dinucleotide was performedbasically as described by T. G. Heckler et al. (Tetrahedron 40: 87-94,1984). Briefly, truncated tRNA molecules (8.6 O.D.₂₆₀ units/ml) andaminoacylated dinucleotides (4.6 O.D.,₂₆₀ units/ml), were incubated with340 units/ml T4 RNA ligase for 16 hours at 4° C. The reaction buffer:included 55 mM. Na-Hepes, pH 7.5, 15 mM MgCl₂, 250 μM ATP, 20 μg/ml BSAand 10% DMSO. After incubation, the reaction mixture was diluted to afinal concentration of 50 mM NaOAc, pH 4.5, containing 10 mM MgCl₂. Theresulting mixture was applied to a DEAE-cellulose column (1 ml),equilibrated with 50 mM NaOAc, pH 4.5, 10 mM MgCl₂, at 4° C. The columnwas washed with 0.25 mM NaCl to remove RNA ligase and other non-tRNAcomponents. The tRNA-containing factions were pooled and loaded onto aBD-cellulose column at 4° C., that had been equilibrated with 50 mMNaOAc, pH 4.5, 10 mM MgCl₂, and 1.0 M NaCl. Unreacted tRNA was removedby washes with 10 ml of the same buffer. Pure misaminoacylated tRNA wasobtained by eluting the column with buffer containing 25% ethanol.

Example 3 Preparation of Extract and Template for Cell-Free Translation.

Preparation of extract: Wheat: germ embryo extract was prepared byfloatation of wheat germs to enrich for embryos using a mixture ofcyclohexane and carbon tetrachloride (1:6), followed by drying overnight(about 14 hours). Floated wheat germ embryos (5 g) were ground in amortar with 5 grams of powdered glass to obtain a fine powder.Extraction medium (Buffer I: 10 mM trisacetate buffer, pH 7.6, 1 nMmagnesium acetate, 90 mM potassium acetate, and 1 mM DTT) was added tosmall portions until a smooth paste was obtained. The homogenatecontaining disrupted embryos and 25 ml of extraction medium wascentrifuged twice at 23,000×g. The extract was applied to a SephadexG-25 fine column and eluted in Buffer II (10 mM trisacetate buffer, pH7.6, 3 mM magnesium acetate, 50 mM potassium acetate, and 1 mM DTT). Abright yellow band migrating in void volume and was collected (S-23) asone ml fractions which were frozen in liquid nitrogen.

Preparation of template: Template DNA was prepared by linearizingplasmid pSP72-bop with EcoRI. Restricted linear template DNA waspurified by phenol extraction and DNA precipitation. Large scale mRNAsynthesis was carried out by in vitro transcription using theSP6-ribomax system (Promega; Madison, Wis.). Purified mRNA was denaturedat 67° C. for 10 minutes immediately prior to use.

Example 4 Cell-Free Translation Reactions.

The incorporation mixture (100 μl) contained 50 μl of S-23 extract, 5 mMmagnesium acetate, 5 mM Tris-acetate, pH 7.6, 20 mM Hepes-KOH buffer, pH7.5; 100 mM potassium acetate, 0.5 mM DTT, 0.375 mM GTP, 2.5 mM ATP, 10mM creatine phosphate, 60 μg/ml creatine kinase, and 100 μg/ml mRNAcontaining the genetic sequence which codes for bacterio-opsin.Misaminoacylated PCB-lysine or coumarin amino acid-tRNA^(tyr) moleculeswere added at 170 μg/ml and concentrations of magnesium ions and ATPwere optimized. The mixture was incubated at 25° C. for one hour.

Example 5 Isolation of Nascent Proteins Containing PCB-Lysine.

Streptavidin coated magnetic Dynabeads M-280 (Dyna; Oslo, Norway),having a binding capacity of 10 μg of biotinylated protein per mg ofbead. Beads at concentrations of 2 mg/ml, were washed at least 3 timesto remove stabilizing BSA. The translation mixture containing PCB-lysineincorporated into nascent protein was mixed with streptavidin coatedbeads and incubated at room temperature for 30 minutes. A magnetic fieldwas applied using a magnetic particle concentrator (MPC) (Dynal; Oslo,Norway) for 0.5-1.0 minute and the supernatant removed with pipettes.The reaction mixture was washed 3 times and the magnetic beads suspendedin 50 μl of water.

Photolysis was carried out in a quartz cuvette using a Black-Raylongwave UV lamp, Model B-100 (UV Products, Inc.; San Gabriel, Calif.).The emission peak intensity was approximately 1100 μW/cm² at 365 nm.Magnetic capture was repeated to remove the beads. Nascent proteinsobtained were quantitated and yields estimated at 70-95%.

Example 6 Determination of the Lower Limit of Detection usingFluorescence.

Bovine serum albumin (BSA), suspended at 0.25 mg/ml in borate buffer, pH8.0, was combined with a 25 fold molar excess fluorescamine (SigmaChemical; St. Louis, Mo.) at 50 mg/ml to produce a modified, fluorescentBSA. Various amounts of modified protein (1 ng, 5 ng, 10 ng, 25 ng, 50ng,r 75 ng, 100 ng, 150 ng, 200 ng) were suspended in loading buffer(bromophenol blue, glycerol, 2-mercaptoethanol, Tris-HCl, pH 6.8, SDS),and added to individual wells of a 1.5 mm thick, 12% polyacrylamide gelwith a 3% stacker. The water cooled gel was electrophoresed for 4 hoursat 50 volts. After electrophoresis, the gel was removed from theelectrophoresis apparatus, placed on a UV transilluminator andphotographed with polaroid Type 667 film using an exposure time of 10seconds. The lowest limit of detection observed under theses conditionswas 10 ng. These results indicate that using equipment found in atypical molecular biology lab, fluorescently labeled proteins can bedetected at ng quantities. Using even more sophisticated detectionprocedures and devices the level of detection can be increased evenfurther.

Example 7 Identification of Nascent Proteins Containing Coumarin-AminoAcid.

Cell-free translation is performed as described using charged tRNA^(tyr)molecules misaminoacylated with lysine coupled to a benzopyrenefluorophore moiety and human γ-interferon mRNA which contains 21 codonsfor lysine. Samples of the mixture are supplemented with buffercontaining bromophenol blue, glycerol, 2-mercaptoethanol, Tris-HCl, pH6.8, and SDS, and directly applied to a 12% poly-acrylarnide gel (3%stacker) along with a set of molecular weight markers. Electrophoresisis performed for 3 hours at 50 volts. The gel is removed from theelectrophoresis apparatus and photographed under UV light. Bands offluorescently labeled interferon protein are specifically detected at amolecular weight of about 25 KDa No other significant fluorescentactivity is observed on the gel. Free misaminoacylated tRNA moleculesmay be electrophoresed off of the gel and not specifically detected.

Example 8 Determination of the In Vivo Half-Life of a PharmaceuticalComposition.

Cell-free translation reactions are performed by mixing 10 μl ofPCB-coumarin amino acid-tRNA^(leu), prepared by chemicalmisaminoacylation as described above and suspended in TE at 1.7 mg/ml),50 μl of S-23 extract 10 μl water and 10 μl of a solution of 50 mMmagnesium acetate, 50 mM Tris-acetate, pH 7.6, 200 mM Hepes-KOH buffer,pH 7.5; 1 M potassium acetate, 5 mM DTT, 3.75 mM GTP, 25 mM ATP, 100 mMcreatine phosphate and 600 μg/ml creatine kinase. This mixture is kepton ice until the addition of 20 μl of 500 μg/ml human IL-2 mRNA(containing 26 leucine codons) transcribed and isolated from recombinantIL-2 cDNA. The mixture is incubated at 25° C. for one hour and placed onice. 100 μl of streptavidin coated magnetic Dynabeads (2 mg/ml) areadded to the mixture which is placed at room temperature for 30 minutes.After incubation, the mixture is centrifuged for 5 minutes in amicrofuge at 3,000×g or, a magnetic field is applied to the solutionusing a MPC. Supernatant is removed and the procedure repeated threetimes with TE. The final washed pellet is resuspended in 50 μl of 50 mMTris-HR, pH 7.5 and transferred to a quartz cuvette. UV light from aBlack-Ray longwave UV lamp is applied to the suspension forapproximately 1 second. A magnetic field is applied to the solution witha MPC for 1.0 minute and the supernatant removed with a pipette. Thesupernatant is sterile filtered and mixed with equal volumes of sterilebuffer containing 50% glycerol 1.8% NaCl and 25 mM sodium bicarbonate.Protein concentration is determined by measuring the O.D.₂₆₀.

0.25 ml of the resulting composition is injected i.v. into the tail veinof 2 Balb/c mice at concentrations of 1 mg/ml. Two control mice are alsoinjected with a comparable volume of buffer. At various time points (0,5 minutes, 15 minutes, 30 minutes, 60 minutes, 2 hours and 6 hours), 100μl serum samples are obtained from foot pads and added to 400 μl of 0.9%saline. Serum sample are added to a solution of dynabeads (2 mg/ml)coated with anti-coumarin antibody and incubated at room temperature for30 minutes. A magnetic field is applied to the solution with a MPC for 1minute and the supernatant removed with a pipette. Fluorescence at 470nm is measured and the samples treated with monoclonal antibody specificfor rat IL-2 protein. IL-2 protein content is quantitated for eachsample and equated with the amount of fluorescence detected. From theresults obtained, in vivo IL-2 half-life is accurately determined.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. It is intended that the specificationand examples be considered exemplary only, with the true scope andspirit of the invention being indicated by the following claims.

1. A conjugate, comprising a substrate covalently coupled to adetectable moiety through a cleavable linker, said substrate selectedfrom the group consisting of nucleosides and nucleotides, saiddetectable moiety having the property of fluorescence.
 2. The conjugateof claim 1, wherein said substrate is a nucleotide selected from thegroup consisting of NTP, dNTP and ddNTP.
 3. The conjugate of claim 2,wherein said nucleotide is part of an oligonucleotide.
 4. The conjugateof claim 1, wherein said detectable moiety comprises coumarin.
 5. Theconjugate of claim 1, wherein said detectable moiety is selected fromthe group consisting of rhodamines and fluoresceins.
 6. A method,comprising: a) providing i) nucleic acid, ii) the conjugate of claim 1,and iii) a polymerase; and b) mixing said nucleic acid and saidpolymerase in the presence of said conjugate under conditions such thatsaid conjugate is incorporated into said nucleic acid to produce labelednucleic acid.
 7. The method of claim 6, wherein said nucleic acid isRNA.
 8. The method of claim 6, wherein said nucleic acid is DNA.